Emergence of within-host SARS-CoV-2 recombinant genome after coinfection by Gamma and Delta variants

Since the first reports of patients coinfected by two genetically-distinct lineages of SARS-CoV-2, the scientific community raised concerns about the recombination of intra-host viral RNA sequences as a possible mechanism underlying the emergence of novel variants. Indeed, this phenomenon occurs at a relatively high frequency among betacoronaviruses. Nevertheless, the few existing studies about recombination between genetically-distinct lineages of SARS-CoV-2 are restricted to detect the inter-host dissemination of genomes post-recombination events. However, the high genomic similarity between the current co-circulating lineages challenges the identification of these events. Here, we report the first case of intra-host SARS-CoV-2 recombination during a coinfection by the variants of concern (VOC) AY.33 (Delta) and P.1 (Gamma) supported by sequencing reads harboring a mosaic of lineage-defining mutations. By using next-generation sequencing reads intersecting regions that simultaneously overlap lineage-defining mutations from Gamma and Delta, we were able to identify a total of six recombinant regions across the SARS-CoV-2 genome within a sample. Four of them mapped in the spike gene and two in the nucleocapsid gene. We detected mosaic reads harboring a combination of lineage-defining mutations from each VOC. To our knowledge, this is the first report of intra-host RNA-RNA recombination between two lineages of SARS-CoV-2, which can represent a threat to public health management during the COVID-19 pandemic due to the possibility of the emergence of viruses with recombinant phenotypes.

Pooling for SARS-CoV2 Surveillance: Validation and Strategy for Implementation in K-12 Schools

Repeated testing of a population is critical for limiting the spread of the SARS-CoV-2 virus and for the safe reopening of educational institutions such as kindergarten—grade 12 (K-12) schools and colleges. Many screening efforts utilize the CDC RT-PCR based assay which targets two regions of the novel Coronavirus nucleocapsid gene. The standard approach of testing each person individually, however, poses a financial burden to these institutions and is therefore a barrier to using testing for re-opening. Pooling samples from multiple individuals into a single test is an attractive alternate approach that promises significant cost savings—however the specificity and sensitivity of such approaches needs to be assessed prior to deployment. To this end, we conducted a pilot study to evaluate the feasibility of analyzing samples in pools of eight by the established RT-PCR assay. Participants (1,576) were recruited from amongst the Tufts University community undergoing regular screening. Each volunteer provided two swabs, one analyzed separately and the other in a pool of eight. Because the positivity rate was very low, we spiked approximately half of the pools with laboratory-generated swabs produced from known positive cases outside the Tufts testing program. The results of pooled tests had 100% correspondence with those of their respective individual tests. We conclude that pooling eight samples does not negatively impact the specificity or sensitivity of the RT-PCR assay and suggest that this approach can be utilized by institutions seeking to reduce surveillance costs.

Analysis of the initial lot of the CDC 2019-Novel Coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel

At the start of the COVID-19 pandemic, the Centers for Disease Control and Prevention (CDC) designed, manufactured, and distributed the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel for SARS-CoV-2 detection. The diagnostic panel targeted three viral nucleocapsid gene loci (N1, N2, and N3 primers and probes) to maximize sensitivity and to provide redundancy for virus detection if mutations occurred. After the first distribution of the diagnostic panel, state public health laboratories reported fluorescent signal in the absence of viral template (false-positive reactivity) for the N3 component and to a lesser extent for N1. This report describes the findings of an internal investigation conducted by the CDC to identify the cause(s) of the N1 and N3 false-positive reactivity. For N1, results demonstrate that contamination with a synthetic template, that occurred while the “bulk” manufactured materials were located in a research lab for quality assessment, was the cause of false reactivity in the first lot. Base pairing between the 3’ end of the N3 probe and the 3’ end of the N3 reverse primer led to amplification of duplex and larger molecules resulting in false reactivity in the N3 assay component. We conclude that flaws in both assay design and handling of the “bulk” material, caused the problems with the first lot of the 2019-nCoV Real-Time RT-PCR Diagnostic Panel. In addition, within this study, we found that the age of the examined diagnostic panel reagents increases the frequency of false positive results for N3. We discuss these findings in the context of improvements to quality control, quality assurance, and assay validation practices that have since been improved at the CDC.

Nucleocapsid (N) Gene Mutations of SARS-CoV-2 Can Affect Real-Time RT-PCR Diagnostic and Impact False-Negative Results

The current COVID-19 pandemic demands massive testing by Real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction), which is considered the gold standard diagnostic test for the detection of the SARS-CoV-2 virus. However, the virus continues to evolve with mutations that lead to phenotypic alterations as higher transmissibility, pathogenicity or vaccine evasion. Another big issue are mutations in the annealing sites of primers and probes of RT-PCR diagnostic kits leading to false-negative results. Therefore, here we identify mutations in the N (Nucleocapsid) gene that affects the use of the GeneFinder COVID-19 Plus RealAmp Kit. We sequenced SARS-CoV-2 genomes from 17 positive samples with no N gene detection but with RDRP (RNA-dependent RNA polymerase) and E (Envelope) genes detection, and observed a set of three different mutations affecting the N detection: a deletion of 18 nucleotides (Del28877-28894), a substitution of GGG to AAC (28881-28883) and a frameshift mutation caused by deletion (Del28877-28878). The last one cause a deletion of six AAs (amino acids) located in the central intrinsic disorder region at protein level. We also found this mutation in 99 of the 14,346 sequenced samples by the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants, demonstrating the circulation of the mutation in Sao Paulo, Brazil. Continuous monitoring and characterization of mutations affecting the annealing sites of primers and probes by genomic surveillance programs are necessary to maintain the effectiveness of the diagnosis of COVID-19.

Establishment of a quantitative RT-PCR detection of SARS-CoV-2 virus

Background The outbreak of novel coronavirus disease 2019 (COVID-19) has become a public health emergency of international concern. Quantitative testing of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus is demanded in evaluating the efficacy of antiviral drugs and vaccines and RT-PCR can be widely deployed in the clinical assay of viral loads. Here, we developed a quantitative RT-PCR method for SARS-CoV-2 virus detection in this study. Methods RT-PCR kits targeting E (envelope) gene, N (nucleocapsid) gene and RdRP (RNA-dependent RNA polymerase) gene of SARS-CoV-2 from Roche Diagnostics were evaluated and E gene kit was employed for quantitative detection of COVID-19 virus using Cobas Z480. Viral load was calculated according to the standard curve established by series dilution of an E-gene RNA standard provided by Tib-Molbiol (a division of Roche Diagnostics). Assay performance was evaluated. Results The performance of the assay is acceptable with limit of detection (LOD) below 10E1 copies/μL and lower limit of quantification (LLOQ) as 10E2 copies/μL. Conclusion A quantitative detection of the COVID-19 virus based on RT-PCR was established.

Longitudinal SARS-CoV-2 RNA Wastewater Monitoring Across a Range of Scales Correlates with Total and Regional COVID-19 Burden in a Well-Defined Urban Population

Wastewater-based epidemiology (WBE) is an emerging surveillance tool that has been used to monitor the ongoing COVID-19 pandemic by tracking SARS-CoV-2 RNA shed into wastewater. WBE was performed to monitor the occurrence and spread of SARS-CoV-2 from three wastewater treatment plants (WWTP) and six neighborhoods in the city of Calgary, Canada (population 1.3 million). A total of 222 WWTP and 192 neighborhood samples were collected from June 2020 to May 2021, encompassing the end of the first-wave (June 2020), the second-wave (November end to December, 2020) and the third-wave of the COVID-19 pandemic (mid-April to May, 2021). Flow-weighted 24-hour composite samples were processed to extract RNA that was then analyzed for two SARS-CoV-2-specific regions of the nucleocapsid gene, N1 and N2, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Using this approach SARS-CoV-2 RNA was detected in 98.06 percent (406/414) of wastewater samples. SARS-CoV-2 RNA abundance was compared to clinically diagnosed COVID-19 cases organized by the three-digit postal code of affected individuals primary residences, enabling correlation analysis at neighborhood, WWTP and city-wide scales. Strong correlations were observed between N1 and N2 gene signals in wastewater and new daily cases for WWTPs and neighborhoods. Similarly, when flow rates at Calgarys three WWTPs were used to normalize observed concentrations of SARS-CoV-2 RNA and combine them into a city-wide signal, this was strongly correlated with regionally diagnosed COVID-19 cases and clinical test percent positivity rate. Linked census data demonstrated disproportionate SARS-CoV-2 in wastewater from areas of the city with lower socioeconomic status and more racialized communities. WBE across a range of urban scales was demonstrated to be an effective mechanism of COVID-19 surveillance.

Development and Evaluation of a Nested PCR for Improved Diagnosis and Genetic Analysis of Peste des Petits Ruminants Virus (PPRV) for Future Use in Nascent PPR Eradication Programme

Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants caused by PPR virus (PPRV). PPR is endemic in Asia, the Middle East and across large areas of Africa and is currently targeted for global eradication by 2030. The virus exists as four different lineages that are usually limited to specific geographical areas. However, recent reports of spread of PPRV, in particular of lineage IV viruses to infection-free countries and previously PPR endemic areas are noteworthy. A rapid and accurate laboratory diagnosis and reports on its epidemiological linkage for virus spread play a major role in the effective control and eradication of the disease. Currently, molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) are usually used for diagnosis of PPR while the sequencing of part of the nucleocapsid gene is usually carried out for the viral lineage identification. However, it is difficult to diagnose and sequence the genetic material if the animal excreted a low level of virus at the initial stage of infection or if the PPRV is degraded during the long-distance transportation of samples to the reference laboratories. This study describes the development of a novel nested RT-PCR assay for the detection of the PPRV nucleic acid by targeting the N-protein gene, compares the performance of the assay with the existing conventional RT-PCR and also provides good-quality DNA suitable for sequencing in order to identify circulating lineages. The assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I–IV) of PPRV. This assay provides a solution with an easy, accurate, rapid and cost-effective PPR diagnostic and partial genome sequencing for use in resource-limited settings.

482. SARS-CoV-2 Prevalence in Feces of Very Young Children, A Longitudinal Study

Background Understanding the disease burden of SARS- CoV-2 in young children has been challenging as the majority are asymptomatic or experience mild symptoms and were rarely tested. SARS-CoV-2 is traditionally detected through respiratory secretions but has also been reported in feces where shedding may continue for weeks after respiratory samples show resolution. We examined the prevalence of SARS-CoV-2 in already collected fecal samples from young children through the pandemic as well as associated demographic factors. Methods As part of an ongoing longitudinal microbiome study in Northern Virginia, serial stools samples were collected from infants before and throughout the Covid-19 pandemic. Reverse transcription quantitative-PCR detecting SARS-CoV-2 nucleocapsid gene in the N1 and N2 regions was performed. Penalized logistic regression models were developed to evaluate the association between fecal positivity and potential risk factors. Results The overall prevalence of SARS-CoV-2 in infant feces was 1.69 % (13 samples) with a prevalence at delivery, 2, 6, 12 and 24 months of 0, 0, 2.56, 1.96, and 0.85 % respectively. Fecal positivity was first detected 31 days before the reported first case of Covid-19 in Northern Virginia; prevalence rates peaked in September at 4.5% (Figure 1). Only one infant who tested positive was symptomatic with COVID-19 21 days before his stool was collected. Of the 13 positive samples, 8 reported Hispanic ethnicity and 7 reported an essential worker (Table 1). Penalized logistic regression model showed association between Hispanic ethnicity and testing positive (OR 5.04 (95% CI 1.7 – 15.0)) that remained after controlling for the presences of an essential worker (OR 4.7 (95% CI 1.6 – 14.0)). Conclusion Prevalence of SARS- CoV-2 in infant stool correlated with the prevalence of COVID-19 during the pandemic, with higher rates in those of Hispanic ethnicity corelating with regional trends. Fecal positivity in asymptomatic infants even before quarantine restrictions supports the early but silent transmission of SARS-CoV-2. This study likely underestimates true prevalence rates as stool samples were stored without viral preservative. There are many socioeconomic factors that predispose to disease while ethnicity may be a mediating or confounding factor Disclosures All Authors: No reported disclosures

RT-qPCR Detection of SARS-CoV-2 from Wastewater Using the AB 7500 v1

This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This protocol describes triplex and duplex assays for the RT-qPCR detection of the nucleocapsid region of the SARS-CoV-2 genome. These assays, along with the murine norovirus (MNV; extraction control) and crAssphage (human indicator) RT-qPCR assay (RT-qPCR Detection of Process Controls (Murine noroviurs and crAssphage) from Wastewater (protocols.io)), were developed for use on the AB 7500 platform using software version 2.0 or 2.3. All assays incorporate an internal amplification control (IC) to prevent the reporting of false negatives due to inhibition or failure of the RT-qPCR. These multiplexed detection assays were developed for the qualitative determination SARS-CoV-2 nucleocapsid gene extracted from wastewater. Valid sample results are contingent upon the detection of the MNV extraction control from the sample being tested.